AIDS (acquired immunodeficiency syndrome) is caused by HIV (Human Immunodeficiency Virus) and is characterized by immunodeficiency, tumors, such as Kaposi's sarcoma (KS) and B-cell lymphomas, opportunistic infections and central nervous system disorders. Since AIDS is spread world-wide and has a high mortality, one of the most important Public Health goal is to develop a prophylactic and/or therapeutic vaccine against HIV or AIDS. Most of the past and current strategies have used the viral envelope or its sub-units as immunogens, but with unsatisfactory results due to the high variability of the viral envelope (Ref. 162, 112—throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which the present invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims). Therefore, as an alternative to sterilizing immunity, it is a common opinion that it could be sufficient to block progression of infection and disease onset. Moreover, immuno-protective responses can be obtained utilizing DNA regions of HIV as immunogens (Ref. 91, 17). Owing to the published experimental data, the inventor believes that it is necessary to produce a vaccine that utilizes viral products other than env. In particular, the viral proteins to be used as immunogens must be more conserved among HIV isolates, capable of inducing an effective immune response, both humoral and cellular, and must have a vital function for the virus. Such products must be experimented in the model of non human primates (because their immune system is more similar to that of humans as compared to phylogenetically more distant animals) and in which AIDS can develop after virus infection.
The HIV-1 Tat protein has all the characteristics to be a good immunogen for vaccine purposes: it is conserved, immunogenic and essential in the early phases of the viral infection. Moreover, Tat has a key role not only in viral replication, transmission and progression of the infection, but also in the onset and progression of AIDS-associated tumors, for instance KS, which is the most frequent AIDS-associated tumor, and of other syndromes and symptoms developing after HIV infection.
Tat is a protein of 86-102 amino acids, depending on the viral strain, coded by two exons. Tat is produced soon after the infection, localizes in the nucleus and transactivates the expression of all viral genes by interacting with the “Tat-responsive element” (TAR) present in the LTR (Ref. 25). Tat has also a role in HIV virulence (Ref. 63, 113, 60, 84). The product of the first exon (amino acids 1-72) is conserved among different viral isolates (Ref. 112) and is sufficient for the transactivation of the HIV-1 products (Ref. 25). It contains 4 domains. The acidic domain (amino acids 1-21) is important for the Tat interaction with cellular proteins; the cysteine rich region (amino acids 22-37) represents the transactivation domain. This region is the most conserved among the primary isolates (Ref. 108) of cysteine 22 with a glycine abolishes the capacity of Tat to transactivate the HIV-LTR (Ref. 166) the core domain (amino acids 38-48) is also conserved and it is important for function. Substitution of lysine 41 with a threonine inactivates the transactivating activity of Tat on the HIV LTR (Ref. 70); the basic domain (amino acids 49-57), rich in arginine and lysine, is necessary for the nuclear localization of Tat and binds specifically its RNA target (TAR) (Ref. 25). Moreover, the basic region is responsible for the binding of extracellular Tat to heparin and to heparansulphate proteoglycans (HSPG) (Ref. 26). Mutations in the basic region abolish such interactions. The carboxy-terminal portion of Tat is not necessary for the LTR transactivation, but contains an arginine-glycine-aspartic acid sequence (RGD), usually present in the extracellular matrix proteins (ECM), that is responsible for the binding of Tat to the integrin receptors α5β1 and αvβ3. These interactions mediate the Tat effects on AIDS-associated tumors and on the immune, vascular and nervous system (Ref 11, 42, 170, 25). During the acute infection of T-cells with HIV-1, or after transfection of the tat gene in COS-1 cells, the Tat protein is released in the absence of cellular death in the extracellular environment (Ref. 40, 41, 25). Tat release from infected cells occurs also in vivo since extracellular Tat is present in the serum of infected subjects (Ref. 164) and in AIDS-KS lesions (Ref. 42). After release, part of the protein remains in a soluble form, and part binds to the HSPG of the ECM. Tat bound to the HSPG can be recovered in a soluble form by the addition of heparin. The binding with heparin is due to the Tat basic region; it prevents the effects of extracellular Tat and protects the protein from oxidation. This feature has been used by us to purify Tat with a high biological activity (Ref. 26). Extracellular Tat can be internalized by cells, can migrate into the nucleus and transactivate viral gene expression (Ref. 49, 98, 100, 41). The internalization of Tat occurs by endocytosis mediated by the binding of RGD region of Tat to α5β1 and αvβ3 (Ref 10, 42, Ensoli et al., unpublished data) and/or by the basic region which binds to HSPG.
Tat can activate viral replication and virus transmission also through indirect mechanisms involving the modulation of the expression of cellular genes which play a key role in the control of cell survival, and on the expression of inflammatory cytokines (IC) with an effect on viral replication (Ref. 25).
Beyond its importance in viral replication, Tat plays an important role in AIDS pathogenesis. Tat is able to modulate the survival and proliferation of infected and non-infected cells by causing activation or repression of cytokines, such as IL-2 (Ref. 123, 163, 31), or of genes with a key role in the cell cycle (Ref. 145, 169, 164, 173). The anti- or pro-apoptotic effects of Tat depend on a number of factors such as the cell type, the fact that Tat is expressed by the cell or added to the cell and on its concentration (Ref. 40, 41, 171).
Tat is the factor responsible for the enhanced frequency and aggressiveness of KS in HIV-1 infected subjects (Ref. 43, 33). KS is a tumor of vascular origin and it is the most frequent neoplasia in HIV-infected individuals. Tat induces KS cells and endothelial cells activated by IC to migrate, to express type IV collagenase, to invade the ECM and to proliferate, such mechanisms being necessary for angiogenesis and tumor invasion (Ref. 40, 41, 42, 2, 46). Such effects of Tat are induced by IC, since they stimulate the expression of the Tat receptors, α5β1 and αvβ3 (Ref 10). Tat mimics the effect of ECM proteins, such as fibronectin and vitronectin and both the RGD region and the basic region are necessary for the effects of the extracellular Tat on KS cells, on angiogenesis and on progression of KS. The capability of extracellular Tat of binding in vivo its receptors in the AIDS-KS lesions (Ref. 40) support the idea that Tat is involved in the onset and the maintenance of AIDS-associated KS. Moreover mice transgenic for the tat gene develop KS or other phenotypes depending on the level of expression of the transgene (Ref. 160, 34).
It has been suggested that Tat plays a role in the hyperproliferative phenomena and in the pathogenesis of the B lymphomas, frequently observed in seropositive subjects and in tat-transgenic mice (Ref. 157), through mechanisms involving the enhancement of bcl-2 and cytokines expression (Ref. 122). Other evidence confirms a probable role of Tat in oncogenesis (Ref. 72).
Tat can also activate the expression of viral promoters, such as those of herpesviruses and of other viruses which reactivate in AIDS individuals, promoting the onset and progression of opportunistic infections (Ref. 25).
Tat seems also able to exert neurotoxic effects both direct (through the basic and the RGD regions) and indirect through induction of IC having a toxic effect on the neurons of the central nervous system or on the hematoencephalic barrier (Ref. 25). Regarding the immune response to Tat, a number a studies suggest that anti-Tat antibodies play a protective role in the control of the evolution of the disease in vivo (Ref. 130, 135, 136, 149, 127). Moreover, in vitro, anti-Tat antibodies not only suppress the internalization, the transcellular activation of Tat and viral infection (Ref. 41, 127), but they also inhibit the proliferation and Tat-induced migration of KS cells and the formation of KS-like lesions in mice (Ref. 40, 41, 42). Finally, our preliminary results demonstrate that anti-Tat antibodies are absent in AIDS-KS subjects, suggesting that such subjects cannot block the activity of extracellular Tat.
The development of an anti-Tat cell-mediated response in the initial phase of infection is important for the control of the infection itself (Ref. 161, 133, 59) and there exists an inverse correlation between the presence of specific anti-Tat CTL and disease progression (Ref. 156). Such results were obtained in studies on macaques inoculated with SIVmac (Ref. 91, 158). Moreover, recent data in mice of different species in which Tat was inoculated either as a plasmid or as protein showed that it is possible to induce both a humoral and cellular response to the protein (Ref. 61). However, it has been observed variability among several mouse species and such results have not been reproduced with the same immunogens in non-human primates (Ref. 124). The lack of reproducibility in non human primate model of the results from vaccine experiments performed in mice is frequent and possibly due to the different immune system of these two species which can raise different immune responses with the same immunogen, as demonstrated for the HIV Env protein. Thus, candidate vaccines for humans must be tested in non-human primates and not only in inferior species.
The inventor believes that other viral proteins, or parts thereof, could be associated with Tat to enhance a specific immune response against HIV and could be of benefit also in the vaccination against the onset of tumors and of other pathologies and symptoms associated with HIV infection. Such products are the Nef, Rev and Gag proteins of HIV.
Nef is another viral regulatory protein important for the development of disease (Ref. 3, 48, 58). Nef is produced early after infection and it is released in the extracellular environment (Ensoli, unpublished data). In the SIVmac/macaque system the presence of Nef correlates with high viral replication and with progression to AIDS (Ref. 71). Nef is more variable than Tat (Ref. 112). Nef is an immunogenic protein (Ref. 53, 32, 35, 151) and it is capable of inducing CTL (Ref. 16, 36). In particular, it has been identified an immunodominant region of Nef (region 73-144) which is recognized by CTLs in most HIV-infected patients.
Rev is a viral regulatory protein produced early during infection (Ref. 51, 119) and released in the extracellular environment (Ensoli et al., unpublished data). Rev is essential for HIV replication and for disease progression, and is coded by two exons, partially overlapping Tat-coding regions. Rev is a nuclear protein (Ref. 44) necessary for the expression of the viral messenger RNAs coding for the late proteins (Ref. 97). Rev is a highly conserved protein among the various viral isolates of HIV-1 (Ref. 111) and it is immunogenic. In fact, it induces the production of specific antibodies directed against the two functional domains of the protein (Ref. 120) during the natural infection in man (Ref. 131) and after inoculation in mice (Ref. 61). Lower levels of anti-Rev antibodies in the sera of infected individuals seem to correlate with the progression to AIDS (Ref. 131). Rev can induce CTL both in man and in monkey (Ref. 156, 158) and it has been reported that a specific anti-Rev CTL response, early during the infection, is inversely correlated with disease progression (Ref. 156, 158).
Another viral target is the gag gene, which is expressed late during infection and codes for a group of highly immunogenic structural proteins of the capsid (Ref. 18, 147). The anti-Gag antibody titers are high and stable during the asymptomatic phase of infection, and reach very low levels when the infection progresses to full-blown AIDS, in combination with the drop of CD4+ lymphocytes and the presence of the virus in the peripheral blood (Ref. 174, 73). Gag proteins induce CTL activity early during infection, both in man and in primates (Ref. 103, 168), and their presence is significantly related with the control of the initial viremia and with disease progression (Ref. 175, 6, 134, 167, 92). Finally, p17 and p24 proteins contain immunodominant epitopes which are maintained in different HIV-1 and HIV-2 isolates and are recognized by CTL (Ref. 89, 19, 114, 155, 115).
The inventor believes that cytokines or parts thereof, such as IL-12 and IL-15, or other immuno-modulant cytokines such as IFNα or IFNβ or other proteins enhancing the immunogenic effect of Tat, can be utilized as adjuvants in the formulation of the anti-Tat vaccine. IL-12 is a strong immunoregulatory cytokine produced by antigen-presenting cells (APC) such as B and dendritic cells (Ref. 154). IL-12 is produced early after HIV infection and has a pro-inflammatory action inducing NK cells and T-lymphocytes to produce IFNγ which activates phagocytes and promotes the induction of Th1 lymphocytes. IL-12 plays a fundamental role in the resistance to a number of infections caused by bacteria, fungi, viruses and shows a high anti-tumor activity. Several evidences suggest that viruses which induce immunosuppression, such as HIV and measles virus, act also through mechanisms which suppress IL-12 production (Ref. 57, 50, 144).
IL-15 is a pleiotropic cytokine expressed by non-lymphoid tissues, by activated monocytes/macrophages and by dendritic cells (DC) (Ref. 125, 66). IL-15 plays an important role in regulating the NK activity, in the proliferation of T lymphocytes and in the CTL activity (Ref. 67, 24). IL-15 induces the expression of CTLs against HIV antigens, in the absence of IL-2 and functional CD4+ T-lymphocytes (Ref. 68, 1). Moreover, similarly to IL-2, IL-15 induces the expansion of lymphocytes with cytotoxic activity (“lymphokine-activated killer”, LAK) and stimulates production of IFNγ in PBMCs of seropositive patients (Ref. 93). IL-15 activates monocytes to produce chemokines, playing a role in the onset of inflammatory processes (Ref. 8).
Recent studies have shown that the co-stimulation of CD4+ lymphocytes with paramagnetic beads, coated with anti-CD3 and anti-CD28 monoclonal antibodies determines a logarithmic and polyclonal expansion of lymphocytes from HIV-infected subjects (Ref. 82) without activating virus replication and transmission. Such antiviral activity is a consequence of both the negative modulation of the expression of CCR5, the co-receptor of HIV-1 monocytotropic strains (Ref. 23) and, to a lesser extent, of the high levels of chemokines induced by the co-stimulation with anti-CD3 and anti-CD28 monoclonal antibodies (Ref. 132). The inventor believes that the possibility to expand autologous lymphocytes from HIV infected subjects in the absence of viral replication/transmission, permits to obtain an effective ex vivo immunization, described in the examples, which can be highly helpful in developing an anti-Tat vaccine.
Within the different systems aimed at the generation of effective antiviral and anti-tumor vaccines, the inventor believes that the utilization of dendritic cells could be key in the induction of an immune response to Tat. This is due to the fact that these cells are the most efficient in presenting the antigen and the sole able to stimulate naive lymphocytes, in the absence of adjuvants (Ref. 150). The use of dendritic cells replaces the function of several adjuvants consisting in the induction of a non specific immune response (natural immunity) which, in turns, generates a strong primary specific response in the presence of the antigen.
Since the transmission of HIV infection primarily occurs at the mucosal level (genital and rectal in the adult, oral in the new-born), the inventor believes that the induction of protective immunity at the mucosal level is a primary goal. Many studies have recently shown the possibility to induce mucosal immunization, local and systemic. Particularly, the nasal and oral routes have shown to be the most efficient in inducing an effective mucosal immune response, even at distant sites, such as the genital mucosa (Ref. 138, 118). In particular, the inventor believes that the use of S. Gordonii and Lactobacillus bacteria, modified to express the above mentioned viral antigens, might be a valid strategy to induce or potentiate a specific immune response at the mucosa level in monkeys and in man. These bacteria are, in fact, able to colonize the mouse oral and vaginal mucosa, and to induce a specific, local and systemic, antibody response against heterologous antigens expressed on the surface of recombinant bacteria (Ref. 116, 104, 106, 121, 117, 139, 105, 107). Finally, these bacteria act as live vectors and can induce a prolonged stimulation of the immune system. Moreover, the inventor believes that non-replicating and non-pathogenic recombinant viral vectors, such as herpes simplex type-1 viruses (HSV-1) (Ref. 99), can be used to express viral proteins for systemic (intradermic) and mucosal (oral, vaginal and nasal routes) immunization. In fact, these vectors can accommodate large exogenous sequences (Ref. 52, 64), such as several HIV genes (regulatory, accessory and structural). Moreover, herpes vectors can also be administered via the oral, nasal or vaginal route (Ref. 176, 75).
The inventor believes that Tat (either as protein or DNA), alone or in combination with the other immunogens described above, can be inoculated also by using new delivery systems, such as erythrocytes or nanoparticles. In particular, the inventor believes that it is possible to deliver antigens bound to the membrane of autologous erythrocytes (Ref. 95, 96). Since these erythrocytes are removed from the blood by macrophages, professional antigen presenting cells, only after 120 days, this feature can be used for vaccine purposes. Finally, another delivery strategy is the use of nanoparticles that can carry proteins and DNA (Ref. 27, 172). Nanospheres are polymeric colloidal particles of diverse chemical composition, variable from 10-1000 nm. Different substances (oligonucleotides, drugs, proteins, peptides, DNA) can be loaded on their surface or absorbed in the particle and delivered into the cytoplasm or the nucleus of the cells from where they are slowly released. This allows the utilization of very small amounts of the substance to be delivered.
Based on the results described above, the inventor believes that the immunization with Tat, alone or in combination with other viral products or immuno-modulant cytokines, or parts thereof, in the presence or not of adjuvants, could block viral replication in subjects exposed after vaccination and in the infected subjects, maintaining the infection in an abortive phase, which can be more easily controlled by the immune system. Therefore, the inventor believes that a Tat-based vaccine should be able to induce an immune response, both humoral and cellular, sufficient to block or reduce the replication or the transmission of the virus and therefore capable of controlling virus replication and of blocking productive infection, progression to disease and the onset of tumors and other AIDS-associated syndromes and symptoms. It is, therefore, possible to use the anti-Tat vaccine for both preventive and therapeutic purposes. In fact, a humoral response against Tat could neutralize the effects of extracellular Tat reducing and limiting the infection, whereas the cell-induced response against Tat as well as against other viral proteins enclosed in the vaccine formulation, could destroy the virus infected cells leading to the control the infection. This allows the necessary period of time to the immune system for developing a complete response towards all viral components of the infecting virus in the absence of irreversible damages due to viral replication.
It has been described the use of Tat as an immunogen (WO 95/31999). However, it is disclosed the use of a biologically inactive protein; moreover, in the same patent application no evidence is shown of the biological activity of the “native” Tat protein.
In addition, there is a strong technical prejudice against the use of a biologically active Tat protein, in that it is believed to enhance viral replication in infected subjects and/or to give immunosuppression in seronegative or seropositive individuals (A. Tonelli: Aids, un vaccino per sperare. “La Repubblica”, pag. 10, 24 Oct. 1998).
As evident from the above, despite the efforts made, an efficacious anti-HIV vaccine based on Tat has not been developed yet.